Biological factors, such as coenzymes, cofactors, vitamins and others, play important roles in biological conversion processes. They are usually produced in small amounts in a whole cell (1xc3x9710xe2x88x925-1xc3x9710xe2x88x926). Isolation and purification of such biological factors from normal cells, however, is a difficult project.
Hyperimmunized eggs have been developed and have been shown to over-produce antibodies and certain biological factors. Because hyperimmunization is performed by injection of polyvalent bacterial antigens into the target animal, the amount of biological factors found in eggs from these hyperimmunized animals can not be increased more than one order of magnitude. Therefore, the small amount of biological factors in hyperimmunized egg makes purification of biological factors difficult, and, as such, there is a need for an efficient process for isolating, purifying or otherwise producing such biological factors.
The normal immune system is under a balance in which proinflammatory and anti-inflammatory cells and molecules are carefully regulated to promote normal host immune defense without the destruction of host""s tissues. Once this careful regulatory balance is disturbed, nonspecific stimulation and activation can lead to increased amounts of potent destructive immunological and inflammatory molecules being produced and released. Thus, excess production of proinflammatory cytokines or production of cytokines in the wrong biological context, are associated with mortality and pathology in a wide range of diseases, such as malaria, sepsis, rheumatoid arthritis, inflammatory bowel disease, cancer and AIDS, among others.
Cytokines are pluripotent polypeptides that act in autocrine/paracrine fashions by binding to specific cellular receptors. Their secretion is important in determining the duration and intensity of an immune response. For example, in mice, distinct subsets of CD4+ T helper (Th) clones secrete what have classically been described as Th1 and Th2 cytokines. Th1 cells produce interleukin-2 (IL-2) and interferon-xcex3 (IFN-xcex3) and facilitate the cellular immune response. Th2 cells produce IL-4, IL-5, IL-6 and IL-10 and support the activation of immunoglobulin secreting cells. During the process of inflammation, cytokines such as IL-1xcex2, IL-6 and tumor necrosis factor-xcex1 (TNFxcex1) are released at the site of inflammation. These cytokines have pleiotropic effects and mediate a wide range of symptoms associated with inflammation.
A key cytokine, TNF-xcex1, also known as cachectin, is a 17 kiloDalton protein composed of 157 amino acids and produced mainly by monocytes and activated macrophages. TNF-xcex1 has been shown to possess tumoricidal activity as well as a variety of physiological effects with most major organ systems. In the central nervous system, TNF-xcex1 is involved in fever, anorexia, and alterations in pituitary hormone release. In the cardiovascular system, TNF-xcex1 plays a role in shock, acute respiratory distress and capillary leakage syndrome (procoagulation). TNF-xcex1 is instrumental in the process of acute tubular necrosis and nephritis in the kidney and ischemia, colitis, and hepatic necrosis in the gastrointestinal system. It is also a key cytokine involved in the process of inflammation.
Various genera of the class Aves, such as chickens (gallus domesticus), turkeys, and ducks, produce antibodies in blood and eggs against immunogens that cause avian diseases, as well as against other immunogens. For example, LeBacq-Verheyden et al. (Immunology 27:683 (974)) and Leslie, G. A., et al. (J. Med. 130:1337 (1969)), have quantitatively analyzed immunoglobulins of the chicken. Polson et al. (Immunological Communications 9:495-514 (1980)) immunized hens against several proteins and natural mixtures of proteins, and detected IgY antibodies in the yolks of the eggs. Fertel et al. (Biochemical and Biophysical Research Communications 102:1028:1033 (1981)) immunized hens against prostaglandins and detected antibodies in the egg yolk. Jensenius et al. (Journal of Immunological Methods 46:63-68 (1981)) provide a method of isolating egg yolk IgG for use in immunodiagnostics. Polson et al. (Immunological Communications 9:475-493 (1980)) describe antibodies isolated from the yolk of hens that were immunized with a variety of plant viruses.
U.S. Pat. No. 4,357,272 discloses the isolation of antibodies from the yolks of eggs derived from hyperimmunized hens. The antibody response was elicited by repetitive injections of immunogens derived from plant viruses, human IgG, tetanus antitoxin, snake antivenins, and Serameba.
U.S. Pat. No. 4,550,019 discloses the isolation from egg yolks of antibodies raised in the hen by hyperimmunization with immunogens having a molecular or particle weight of at least 30,000. The immunogens used to hyperimmunize the chickens were selected from among plant viruses, human immunoglobulins, tetanus toxin, and snake venoms.
U.S. Pat. No. 4,748,018 discloses a method of passive immunization of a mammal that comprises parenterally administering purified antibody obtained from the eggs of an avian that has been immunized against the corresponding antigen, and wherein the mammal has acquired immunity to the eggs.
U.S. Pat. No. 5,772,999, assigned to DCV-Biologics, discloses a method of preventing, countering or reducing chronic gastrointestinal disorders or Non-Steroidal Anti-Inflammatory Drug-induced (NSAID-induced) gastrointestinal damage in a subject by administering hyperimmunized egg and/or milk or fractions thereof to the subject.
The present invention is based on the present inventors"" discovery that there is specific immunoregulatory activity in egg, and particularly in egg obtained from hyperimmunized animals, which when administered to a subject animal, and particularly, to mammals, activates cytokine production in that subject animal.
More specifically, the present invention is directed to a highly purified Cytokine Activating Factor which can be obtained from the eggs of an avian. The Cytokine Activating Factor can be highly purified from fractions isolated from both egg yolk and egg white. The Cytokine Activating Factor can also be produced recombinantly or by chemical synthesis.
One embodiment of the present invention relates to an isolated Cytokine Activating Factor (CAF) protein. Such protein comprises an amino acid sequence selected from the group of: (a) an amino acid sequence selected from the group of SEQ ID NO:1 and SEQ ID NO:6; and, (b) an amino acid sequence comprising at least 9 consecutive amino acid residues of either of the amino acid sequences of (a). The isolated CAF protein of the present invention upregulates expression of tumor necrosis factor xcex1 (TNFxcex1), interleukin-1xcex2 (IL-1xcex2) and/or interleukin-6 (IL-6), and/or downregulates expression of transforming growth factor xcex2 (TGFxcex2). In a preferred embodiment, the isolated protein of the present invention comprises an amino acid sequence having at least about 15 consecutive amino acid residues of either of the amino acid sequences of (a), and more preferably, at least about 20 consecutive amino acid residues, and even more preferably, at least about 25 consecutive amino acid residues of either of the amino acid sequences of (a). In another embodiment, the isolated protein of the present invention comprises an amino acid sequence having at least about 50 consecutive amino acid residues of SEQ ID NO:6.
In one embodiment of the present invention, an isolated protein of the present invention comprises an amino acid sequence that is at least about 65% identical to an amino acid sequence of (a) over at least 15 amino acids of the amino acid sequence of (a). Preferably, the protein comprises an amino acid sequence that is at least about 75% identical to an amino acid sequence of (a) over at least 15 amino acids of the amino acid sequence of (a). In another embodiment, the protein comprises an amino acid sequence that is at least about 60% identical to SEQ ID NO:6 over 66 amino acids of SEQ ID NO:6. In yet another embodiment, the protein is encoded by a nucleic acid sequence that hybridizes under high stringency conditions to a nucleic acid sequence encoding an amino acid sequence selected from the group of SEQ ID NO:1 and SEQ ID NO:6. In a most preferred embodiment, the isolated CAF protein of the present invention comprises amino acid sequence selected from the group consisting of SEQ ID NO:1 and SEQ ID NO:6, and most preferably, SEQ ID NO:6.
In one embodiment, an isolated CAF protein of the present invention has one or more of the following biochemical characteristics: (a) has at least one biologically active subunit which passes through a 3000 MW cut-of f filter; (b) is stable at a temperature up to at least about 50xc2x0 C.; (c) is stable at pH of from about 2 to about 10; (d) is water soluble; (e) is non-steroidal; (f) is negatively charged; (g) is substantially non-polar; and/or, (h) has a xcexmax at about 254 nm. An isolated protein of the present invention is biologically active when administered orally. An isolated protein of the present invention is naturally present in both the egg white and egg yolk of avian eggs. As discussed above, an isolated CAF protein of the present invention preferably upregulates expression of tumor necrosis factor xcex1 (TNFxcex1), interleukin-1xcex2 (IL-1xcex2) and/or interleukin-6 (IL-6). In one embodiment, an isolated protein of the present invention downregulates the expression of transforming growth factor xcex2 (TGFxcex2).
One embodiment of the present invention relates to an isolated antibody that selectively binds to the isolated CAF protein of the present invention.
Yet another embodiment of the present invention relates to a composition. Such composition includes a pharmaceutically acceptable carrier and a cytokine activating factor (CAF) protein of the present invention as described above. In one embodiment, the pharmaceutically acceptable carrier is a food product selected from the group of: (a) a hyperimmune egg product which is selected to be enriched for the CAF protein; and, (b) a food product produced with at least a fraction of a hyperimmune egg product, wherein the fraction comprises an enriched amount of the CAF protein as compared to the hyperimmune egg product. Preferably, the pharmaceutically acceptable carrier comprises a fraction of a hyperimmune egg product containing an enriched amount of the CAF protein as compared to the hyperimmune egg product. Suitable fractions of a hyperimmune egg product include, but are not limited to: liquid egg yolk, liquid egg white, powdered egg yolk, powdered egg white, and a water soluble fraction of the hyperimmune egg product.
In one embodiment, the composition of the present invention is in a form selected from the group of a liquid, an aerosol, a capsule, a tablet, a pill, a powder, a gel and a granule. In another embodiment, the pharmaceutically acceptable carrier comprises a controlled release formulation. In yet another embodiment, the pharmaceutically acceptable carrier is selected from the group of: water, phosphate buffered saline, Ringer""s solution, dextrose solution, serum-containing solutions, Hank""s solution, other aqueous physiologically balanced solutions, oils, esters, glycols, biocompatible polymers, polymeric matrices, capsules, microcapsules, microparticles, bolus preparations, osmotic pumps, diffusion devices, liposomes, lipospheres, cells, and cellular membranes.
Yet another embodiment of the present invention relates to a method to regulate an immune response in an animal. Such a method includes the step of administering to the animal a composition comprising a cytokine activating factor (CAF) protein of the present invention as set forth previously herein. Preferably, the composition includes a pharmaceutically acceptable carrier. In one aspect, the composition is administered at a dose of from about 1 nanogram to about 400 milligrams of the CAF protein per kilogram body weight of the animal. Preferred routes of administration include, but are not limited to: oral, intravenous administration, intraperitoneal administration, intramuscular administration, subcutaneous administration, transdermal delivery, intratracheal administration, inhalation, impregnation of a catheter, by suppository, and direct injection into a tissue. In one embodiment, the composition comprises a food product containing the CAF protein. In a preferred embodiment, the animal is a mammal.
Preferably, administration of the composition upregulates expression of tumor necrosis factor xcex1 (TNFxcex1), interleukin-1xcex2 (IL-1xcex2) and/or interleukin-6 (IL-6) by cells of the animal. In one aspect, administration of the composition downregulates expression of transforming growth factor xcex2 (TGFxcex2) by cells of the animal.
Another embodiment of the present invention relates to a method of treating cancer in an animal. Such a method includes the steps of administering to an animal that has or is at risk of developing cancer a composition comprising a cytokine activating factor (CAF) protein of the present invention as previously described herein. Preferably, the composition is administered at a dose of from about 1 nanogram to about 400 milligrams of the CAF protein per kilogram body weight of the animal. Preferred routes of administration include, but are not limited to: oral, intravenous administration, intraperitoneal administration, intramuscular administration, subcutaneous administration, transdermal delivery, intratracheal administration, inhalation, impregnation of a catheter, by suppository, and direct injection into a tissue at or adjacent to the cancer. In one embodiment, the composition comprises a food product containing the CAF protein.
Preferably, administration of the composition produces a result selected from the group of: reduction in symptoms of the cancer, reduction of a tumor associated with the cancer, elimination of a tumor associated with the cancer, prevention of metastatic cancer, prevention of the cancer and stimulation of effector cell immunity against the cancer.
Yet another embodiment of the present invention relates to an isolated nucleic acid molecule. Such a nucleic acid molecule comprises a nucleic acid sequence selected from the group of: (a) a nucleic acid sequence encoding a protein comprising an amino acid sequence selected from the group consisting of: (i) an amino acid sequence selected from the group consisting of SEQ ID NO:1 and SEQ ID NO:6; and, (ii) an amino acid sequence comprising at least 9 consecutive amino acid residues of an amino acid sequence of (i); and, (b) a nucleic acid sequence that is fully complementary to the nucleic acid sequence of (a). Preferably, the protein encoded by the nucleic acid sequence of (a) upregulates expression of tumor necrosis factor xcex1 (TNFxcex1), interleukin-1xcex2 (IL-1xcex2) and/or interleukin-6 (IL-6). In a preferred embodiment, the nucleic acid molecule comprises a nucleic acid sequence encoding an amino acid sequence selected from the group of SEQ ID NO:1 and SEQ ID NO:6.
One embodiment of the present invention relates to a recombinant nucleic acid molecule comprising an isolated nucleic acid molecule of the present invention as set forth previously herein. Yet another embodiment of the present invention relates to a recombinant cell comprising an isolated nucleic acid molecule of the present invention as set forth previously herein, wherein the cell expresses the nucleic acid molecule. Yet another embodiment of the present invention relates to an isolated nucleic acid molecule of the present invention as set forth previously herein.